The goal of this project is to understand the molecular principles underlying protein binding sites for Ca2+ and Mg2+, especially the principles underlying the ion binding affinity, specificity, and kinetics of these metal sites. Four proteins will be studied. Two proteins provide representative Ca2+ sites of the EF-hand class: calmodulin of D. melanogaster, and the D-galactose receptor of E. coli. The two other proteins provide typical Mg2+ sites of the 'carboxylate cluster' class: the motor regulator protein of E. coli, and the RNase H enzyme of E. coli. The specific aims are to quantitate the ion binding parameters of each representative site, then to develop models relating these parameters to the known site structure. In order to test the models, altered sites will be generated by protein engineering, then the ion binding parameters of these sites will be measured to ascertain whether the predicted changes in parameters have been achieved. Target parameters include the dissociation constants of metal ions possessing different ionic radii and charges, the numbers of protons released by metal binding, and the on- and off-rate constants of ion binding and dissociation, respectively. A better understanding of the molecular mechanisms underlying Ca2+ and Mg2+ binding has important implications for the large number of cellular pathways regulated by these ions. In addition, such knowledge will facilitate the engineering of Ca2+ and Mg2+ sites for use in biosensors, stabilization of protein structure, metal-triggered on/off switches, and regulation of multi-enzyme pathways. An example illustrating the latter two applications will be developed.